Metagenomics and Metatranscriptomics Suggest Pathways of 3-Chloroaniline Degradation in Wastewater Reactors

Biological wastewater treatment systems are often affected by major shifts in influent quality, including the input of various toxic chemicals. Yet the mechanisms underlying adaptation of activated sludge process performance when challenged with a sustained toxin input are rarely studied in a controlled and replicated experimental setting. Three replicate bench-scale bioreactors were subjected to a chemical disturbance in the form of 3-chloroaniline (3-CA) over 132 days, after an acclimation period of 58 days, while three control reactors received no 3-CA input. Nitrification was initially affected by 3-CA but the microbial communities in all three treatment reactors adapted to biologically degrade 3-CA within three weeks of the experiment, resulting in partial nitrification recovery. Combining process and microbial community data from amplicon sequencing with potential functions gleaned from assembled metagenomics and metatranscriptomics data, two putative degradation pathways for 3-CA were identified. The first pathway proceeds via a phenol monooxygenase followed by ortho-cleavage of the aromatic ring, and the second one involves a benzoate dioxygenase and subsequent meta-cleavage of the aromatic ring. The genera Gemmatimonas, OLB8, and Taibaiella correlated significantly with 3-CA degradation. Metagenome-assembled genome data also showed the genus OLB8 to be differentially enriched in treatment reactors, making it a strong candidate as 3-CA degrader. Using replicated reactors, this study has demonstrated the impact of a sustained stress on the activated sludge community and processes carried out by its members, followed by process recovery. By a combination of techniques, we showed that microbial communities can develop degradative capacity following a sustained xenobiotic input, and that targeted culture-independent approaches can suggest plausible mechanisms for 3-CA degradation and identify the taxa potentially contributing to it.